Hydrocarbon activation and mutation induction in the L5178Y mouse lymphoma mutation assay were studied as a function of the concentration of an exogenously added rat liver homogenate activation system (S20). Four polycyclic aromatic hydrocarbons were tested at constant dosage levels with 4 concentrations of S20 for ability to induce forward mutations at the thymidine kinase (TK) locus. With all 4 hydrocarbons, increasing S20 concentration resulted in a significant ( p < 0.001) decrease in mutation frequency and corresponding increase in survival. In the absence of S20, no significant mutation induction was observed. Within 5 min after the addition of S20 to a reaction mixture containing [ 3 H]benzo[a] pyrene[B(a)P] (3 μ g/ml) and appropriate cofactors, 70% of the B(a)P was metabolized with a high S20 concentration, whereas only 14% was metabolized with a low S20 concentration. In the absence of S20, the cells did not metabolize B(a)P. Incubation of B(a)P with a low concentration of S20 in the presence of calf thymus DNA (1.5 mg/ml) resulted in twice as much covalently bound B(a)P per mg DNA (33 pmoles/mg) as incubation with a high concentration of S20 (15 pmoles/mg). The amount of the major B(a)P-DNA adduct, (±)-7 β, 8 α-dihydroxy-9 α,10 α-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene- deoxyguanosine, per mg DNA was 5 times greater with low S20 concentration than with high (11.6 pmoles/mg and 2.1 pmoles/mg, respectively). These results suggest that the decrease in mutation frequency with high S20 concentrations results from the rapid oxidation of the hydrocarbon which reduces the opportunity for mutagenic derivatives that are formed from B(a)P to interact with DNA and induce mutation.